1. Field of the Invention
The present invention relates to a human tissue plasminogen activator variant which is useful in the treatment of thrombosis; a DNA sequence encoding the said plasminogen activator; an expression vector comprising the said DNA sequence; a transformant into which the said vector has been introduced; and a method for producing the said plasminogen activator.
2. Description of the Prior Art
Concerning mature human tissue plasminogen activator (hereinafter, referred to as TPA), particularly detailed research has been devoted to TPA secreted by human melanoma (i.e., Bowes melanoma) cells, and TPA is known to be a glycoprotein comprising 572 amino acid residues [Pennica, D., et al., Nature, 301, 214 (1983)].
TPA is an enzyme which acts upon the plasminogen which is present in the blood and converts this plasminogen into plasmin. The plasmin dissolves the fibrin which causes thrombi. TPA possesses strong affinity with fibrin, moreover, the activity of TPA is fibrin-dependent, hence, TPA is regarded as acting specifically upon thrombi. Therefore, TPA is used therapeutically for the treatment of various types of thrombosis [Grossbard, E. B., Pharmaceutical Research, 4, 375 (1987)]. However, when TPA is administered into the blood stream for the treatment of thrombosis, long-sustained effect is not obtained, and the TPA rapidly clears from the circulation, which constitutes a problem in therapeutic applications. TPA is believed to be metabolized principally in the liver [Fuchs, H. E., et al., Blood, 65, 539 (1985)]. The half-life of TPA in the blood is only about 2 minutes [Collen, D., et al., Circulation, 72, 384 (1985)]. Therefore, rather large quantities of protein are currently administered by continuous infusion to maintain therapeutic plasma levels. A TPA with longer in vivo half-life might permit a single bolus injection, the use of lower doses, and thereby, lower cost for the treatment.
With a view to solving these problems, various types of TPA variants, with longer half-life in vivo obtained by chemical or enzymatic modification or by genetic engineering methods, have been reported in the literature [Browne, M. J., et al., J. Biol. Chem., 263, 1599 (1988); Dodd, I., et al., Thrombosis and Haemostasis, 59, 523 (1988); Kayan, N. K., et al., J. Biol. Chem., 263, 3971 (1988)]. However, although greatly improved with respect to plasma half-life in vivo, the TPA variants described in these reports have proved to be extremely inferior to the TPA with regard to the affinity with fibrin that is one of the characteristic properties of TPA [Larsen, G. R., et al., J. Biol. Chem., 263, 1023 (1988)]. Furthermore, in some case, these TPA variants also display a pronounced decrease in fibrinolytic ability [Hansen, L., et al., J. Biol. Chem., 263, 15713 (1988)].
Thus, TPA variants with superior therapeutic efficacy have not yet been obtained. Therefore, there has existed a need for the development of TPA variants which permit effective treatment of thrombosis with small doses by virtue of slow clearance; retain the original biochemical characteristics of TPA to the greatest possible extent; and cause no significant increases in hemorrhagic or other adverse side effects.
TPA is composed of five regions, i.e., a finger region, a growth factor region, a kringle 1 region, a kringle 2 region, and a region possessing serine protease activity. These regions are aligned in the above-mentioned order from the N-terminus of the amino acid sequence of TPA (Pennica, D., et. al., Nature, 301, 214, supra ). Many of the TPA variants with longer half-life in vivo created thus far, lack the finger region alone, or lack an extensive region including finger region and other regions [Hansen, L., et al., J. Biol. Chem., 263, 15713, supra; Larsen, G. R., et al., J. Biol. Chem., 263, 1023, supra; and Collen, D. , et al. , Blood, 71, 219 (1988)]. These types of TPA variants, lacking the finger region, are improved with respect to persistence in the blood, but display markedly decreased thrombolytic ability. Therefore, plasminogen activation and fibrolytic properties are believed to arise from the finger region.
The aforesaid kringle regions have also been studied. The kringle 2 region is said to be concerned in the affinity of TPA for fibrin and the property of activation of TPA by fibrin. On the other hand, although derivatives lacking the kringle 1 region have been created and studied, the function of this region has not yet been adequately clarified [Zonneveld, A. J. V., et al., Journal of Biological Chemistry, 261, 14214 (1986); Proc. N. A. S., 83, 169 (1986) ].